Effectiveness of eugenol sedation to reduce the metabolic rates of cool and warm water fish at high loading densities

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1 Aquculture Reserch, 214, 1 9 doi:1.1111/re Effectiveness of eugenol sedtion to reduce the metolic rtes of cool nd wrm wter fish t high loding densities Aron R Cupp 1,2, Christopher F Hrtle 2, Kim T Fredricks 1,3 & Mrk P Gikowski 1 1 US Geologicl Survey, Upper Midwest Environmentl Sciences Center, L Crosse, WI, USA 2 University of Wisconsin Stevens Point, Stevens Point, WI, USA 3 Vitero University, L Crosse, WI, USA Correspondence: A R Cupp, US Geologicl Survey, Upper Midwest Environmentl Sciences Center, 263 Fnt Reed Rd., L Crosse, WI 5463, USA. E-mil: cupp@usgs.gov Astrct Effects of eugenol (AQUI-S â 2E, 1% ctive eugenol) sedtion on cool wter, yellow perch Perc flvescens (Mitchill), nd wrm wter, Nile tilpi Oreochromis niloticus L. fish metolic rtes were ssessed. Both species were exposed to, 1, 2 nd 3 mg L 1 eugenol using sttic respirometry. In 17 C wter nd loding densities of 6, 12 nd 24 g L 1, yellow perch controls ( mg L 1 eugenol) hd metolic rtes of mg O 2 kg 1 h 1, while yellow perch exposed to 2 nd 3 mg L 1 eugenol hd significntly reduced metolic rtes of nd mg O 2 kg 1 h 1 respectively. Nile tilpi exposed to 3 mg L 1 eugenol hd significntly reduced metolic rte ( mg O 2 kg 1 h 1 ) reltive to the mg L 1 eugenol control ( mg O 2 kg 1 h 1 ) t loding density of 12 g L 1 in 22 C wter. No significnt differences in metolic rtes for Nile tilpi were found t 24 or 36 g L 1 loding densities when exposed to eugenol. Results suggest tht eugenol sedtion my enefit yellow perch welfre t high densities (e.g. live trnsport) due to reduction in metolic rtes, while further reserch is needed to ssess the enefits of eugenol sedtion on Nile tilpi t high loding densities. Keywords: sedtives, eugenol, respirometry, metolic rte, fish trnsport, loding density Introduction Minimizing metolic disturnce when fish re held t high densities (e.g. live trnsport) cn e chllenging for fish culturists nd is importnt for the welfre nd survivl of fish (Hrmon 9). Mesuring metolic rtes of fish cn e used s n indictor for the qulity of the culture or trnsport environment (Cech 199). Specificlly, chnges in metolic rte cn indicte degrding wter qulity, stress nd physicl injury. Metolic rte (e.g. oxygen consumption) is typiclly mesured in respirometry systems during periods of rest, routine swimming or elevted swimming (Bemish 1963). During intensive culture or fish trnsport, oxygen consumption cn increse due to stress ssocited with tnk loding (Wedemeyer 1972), high loding densities (De Areu, Snri-Ocho, Gonclves & Urinti 8) nd decresed wter qulity (Hill & Forster 4; Crneiro, Kiseler, Swrofsky & Bldisserotto 9). Incresed oxygen consumption is secondry response to physicl or chemicl stressor (Brton & Iwm 1991; Cho & Heth ; Portz, Woodley & Cech 6) nd chronic or multiple cute exposures to stressor cn result in compromised immune system nd ultimtely mortlity (Wendelr 1997). Mortlity resulting from the stress of high loding densities (i.e. trnsport) hs een documented up to 14 dys post trnsport (Ross & Ross 8). Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA. 1

2 Effects of eugenol on metolic rtes A R Cupp et l. Aquculture Reserch, 214, 1 9 To mitigte stressors, sedtives hve een studied s n dditive during trnsport of live fish (Cooke, Suski, Ostrnd, Tufts & Whl 4). Tricinemethnesulfonte [3-minoenzoic cid ethyl ester methnesulfonte (MS-222)] is the only sedtive pproved for finfish use in the United Sttes. MS- 222, n efficcious immoilizing gent (Trushenski, Bowker, Guse & Mullign 212), is restricted in the United Sttes y the 21-dy withdrwl period ssigned y the US Food nd Drug Administrtion (FDA). For fish trnsport, there is strong need for short (<1 dy) withdrwl sedtive tht cn e used in quculture nd y fisheries mngers (Bowker & Trushenski 212). Currently, AQUI- S â 2E (1% ctive eugenol; AQUI-S New Zelnd Ltd., Lower Hutt, New Zelnd) is eing evluted y the FDA to sedte fish to hndlele followed y immedite relese to freshwter. Eugenol (2-methoxy-4-prop-2-enyl-phenol) is the primry ctive ingredient in clove oil (7 9% y weight) nd hs een hevily reserched s generl sedtive of fish (Hoskonen & Pirhonen 4; Rouch, Gomes, Fonsec & Vl 5; Plic, Herolt, Andresen, Menzel & Roth 6; Prk, Im, Seol & Prk 9). Results of vrious studies using clove oil hve shown short induction nd recovery times (Anderson, McKinley & Colvecchi 1997; Gomes, Chves, Becker & Bldisserotto 211), low physiologicl disturnces (Cho & Heth ), rpid sorption nd elimintion from tissues (Kilde, Alln & Kerney 4; Guenette, Uhlnd, Helie, Beudry & Vchon 7) nd the ility to minimize primry nd secondry stress responses (Iverson, Finstd, McKinley & Elissen 3; Deriggi, Inoue & Mores 6; Plic et l. 6). Although eugenol in the rw form of clove oil hs een evluted for multiple properties of sedtion on individul fish, there is gp of informtion concerning how high densities of fish respond to sedtion. Furthermore, few studies hve ssessed product where eugenol is the only ctive ingredient, which is importnt due to the potentil crcinogenic properties of the dditionl ctive ingredients of clove oil (USDHHS 7). Yellow perch Perc flvescens (Mitchill) nd Nile tilpi Oreochromis niloticus L. re two economiclly importnt fish nd were used s model cool nd wrm wter species to monitor metolic rtes. Worldwide, production of fish ctegorized under the common nme tilpi generted over $3.7 illion USD in 9 (FAO 9). In the United Sttes, Nile tilpi re predominntly cultured s food fish, while yellow perch re rised for oth stocking (i.e. recretionl hrvest) nd s food fish (Mlison ; El-Syed 6). Nile tilpi is roust to hypoxic environments, which hs promoted their success in culture worldwide (Adel-Mgid & Biker 1975). However, low dissolved oxygen (i.e. <3.5 mg L 1 ) hs een shown to negtively impct the survivl of yellow perch during intensive nd extensive culture (Mlison ). The ojective of this study ws to exmine the effectiveness of eugenol to reduce the metolic rtes of yellow perch nd Nile tilpi held t high loding densities. Specificlly, identifying eugenol concentrtions tht reduced metolic rtes would provide fish culturists with tool to promote the welfre of fish held t high densities. Mterils nd methods Study nimls Yellow perch [ g wet weight, cm totl length (TL)] were htched in outdoor ponds nd rered in 17 C flow-through tnks t the Upper Midwest Environmentl Sciences Center [(UMESC), L Crosse, WI, USA]. Nile tilpi ( g wet weight, cm TL) were otined from commercil frm in Florid, which dministered feed contining 17methyltestosterone to the fry to produce popultion of >8% phenotypic mles efore shipment to UMESC. Nile tilpi were then rered t UMESC in 24 C flow-through tnks. After the grow-out phse t UMESC, yellow perch nd Nile tilpi were then trnsported in truck tnks to the quculture fcility t the University of Wisconsin Stevens Point (Stevens Point, WI, USA). Yellow perch were held in 28 L nd Nile tilpi in 189 L recirculting quculture systems (RAS) nd fed dily mintennce rtions of ~1% ody weight (BW) per dy. Yellow perch were fed n extruded slmon diet [slow-sinking crumles (1.6 mm); Skretting USA, Tooele, UT, USA] nd Nile tilpi were fed tilpi diet [extruded pellets (6.4 mm); Purin Mills, St. Louis, MO, USA]. Fish were not fed h prior to eugenol exposure. Loding density clcultions Multiple loding densities of yellow perch nd Nile tilpi were exposed to multiple eugenol 2 Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9

3 Aquculture Reserch, 214, 1 9 Effects of eugenol on metolic rtes A R Cupp et l. concentrtions to determine effects on metolic rtes. The totl flsk volume of 1.1 L nd wter displcement of 1. ml g 1 BW were ccounted for in loding density clcultions. Initilly, yellow perch were to e tested t higher loding densities of 12, 24 nd 36 g L 1 nd Nile tilpi t 24, 36 nd 48 g L 1. Due to the conicl shpe of the flsk, it ws determined during pilot trils tht yellow perch t 36 g L 1 nd Nile tilpi t 48 g L 1 would e n unresonle density to fit inside the chmer. Therefore, yellow perch were tested t trget loding densities of 6, 12 nd 24 g L 1 nd Nile tilpi t 12, 24 nd 36 g L 1. Fish weights were lter used to clculte the exct loding densities tested. In ddition, to further explore the effects of eugenol elow the three previous loding densities, low loding density of ~2 g L 1 (yellow perch) nd 4 g L 1 (Nile tilpi) ws tested. This loding density ws sustntilly lower thn the three previous densities nd the results were importnt for ssessing the effects of eugenol on metolic rtes when sedting low densities of fish (e.g. generl sedtion). AQUI-S â 2E clcultion The mss of AQUI-S â 2E required to mke the stock solutions (e.g. 1 mg L 1 eugenol = mg L 1 AQUI-S â 2E) ws clculted using the following eqution: A = [(B * C)/1 3 ]/1% ctive eugenol A = AQUI-S â 2E (g) B = Volume of stock solution (L) C = Trget eugenol concentrtion (mg L 1 ) Respirometry trils AQUI-S â 2E ws weighed directly into 5-mL eker (Mettler Toledo Model AB265-S/FACT lnce, Columus, OH, USA). All stock solutions were prepred y dding the known mss of AQUI-S â 2E to 1 L of wter from the RAS tnk followed y hnd mixing for 1 min. Stock solutions of, 1, 2 nd 3 mg L 1 eugenol were individully prepred. A hndheld ph meter (YSI Model 63; YSI Inc., Yellow Springs, OH, USA) ws used to mesure ph of the stock solution. Stock solutions were used to fill the respirometry chmer, which ws the experimentl unit for the study. Blown ir ws supplied directly into the chmer using n ir pump (Penn-Plx Silent-AirX4 TM ; Pentir Ltd., Minnepolis, MN, USA) with terminl ir diffuser. Fish were group weighed into different loding densities (Ohus Explorer â Model EX422 lnce; Ohus Corp., Prsippny, NJ, USA). Air ws uled into the respirometry chmer until dissolved oxygen redings stilized ner % sturtion. Once stilized, the ir diffuser ws removed nd fish were dded to the chmer. The ruer stopper contining the temperture nd dissolved oxygen proes (YSI Model 535; Xylem Inc., Yellow Springs, OH, USA) ws plced in the flsk to sel the wter from contct with the tmosphere nd it ws ensured tht the flsk ws void of ny ir ules, which would potentilly interfere with dissolved oxygen mesurements. The flsk, contining 2.5 cm mgnetic stir r, ws plced on top of stir plte (Thermolyne Cimrec â 2 Model S46725; Thermo Scientific, Lenex, KS, USA) which ws set to slow, stedy spin. Proes were connected to YSI Model 53 Biologicl Oxygen Monitor (Xylem Inc.) nd HOBO â 4-Chnnel Externl dt logger (ONSET Inc., Bourne, MA, USA). The dt logger recorded temperture nd per cent sturtion outputs from the oxygen monitor. Due to the higher metolic rtes of Nile tilpi, oxygen redings were recorded every 1 s for Nile tilpi nd every 15 s for yellow perch. Fish entered the chmer t % sturtion nd remined in the chmer until dissolved oxygen dropped to ~5 6% sturtion, which rnged from 2 to 12 min y tretment. Wet weight (g) nd TL (cm) were recorded for ll fish. Wet weights for ech tretment group were dded together for use in mss-specific metolic rte nd loding density clcultions. Survivl ws not ssessed post exposure, however ll fish were live when the finl wet weight nd TL were mesured. Metolic rte clcultion Mss-specific metolic rtes were clculted for ech species of fish nd for ech comintion of loding density nd eugenol concentrtion (Cech 199). Per cent sturtion of oxygen ws converted to mg L 1 dissolved oxygen prior to clculting metolic rtes (USGS 211). Clcultions were conducted s follows: MR = [(DO initil DO finl ) 9 V] (T 9 M) MR = metolic rte (mg O 2 kg 1 h 1 ) DO initil = dissolved oxygen concentrtion t n erly point in experiment (mg L 1 ) Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9 3

4 Effects of eugenol on metolic rtes A R Cupp et l. Aquculture Reserch, 214, 1 9 DO finl = dissolved oxygen concentrtion t lte point in the experiment (mg L 1 ) V = volume of chmer (L) T = time etween initil nd finl (h) M = mss of fish (kg) Sttisticl nlyses Dt in tles were reported s men SD. Dt presented grphiclly were expressed s men SEM. Homogeneity of vrinces ws tested using Brtlett s test. Normlity of dt ws tested using the Shpiro Wilk test. A log-trnsformtion ws used to normlize dt when the ssumption of normlity ws invlidted. A one-wy ANOVA nd Tukey s HSD post hoc test were used to test for significnt differences in metolic rtes for ech loding density nd eugenol concentrtion. In cses where trnsformtions did not normlize the dt, the non-prmetric Kruskl Wllis test ws used to test significnce. Anlyses were performed using R (R Core Tem 213; 64-it, version ) with the R Commnder interfce (Fox 5); significnce ws declred when P <.5. Results The men wet weight, TL nd wter qulity prmeters (temperture nd ph) re summrized in Tle 1. Fish weights from ech tretment group were used to clculte the loding densities of ech replicte (Tle 2). Yellow perch 6, 12 nd 24 g L 1 loding densities The three loding densities were tested independently t four eugenol concentrtions (Fig. 1). Significnt differences in metolic rtes were detected Tle 1 Men (SD) totl length (cm), wet weight (g) nd wter qulity prmeters (temperture nd ph). Yellow perch hd only one ph mesurement therefore no vrince estimtor ws reported Totl length (cm) Wet weight (g) Temperture ( C) ph for yellow perch exposed to, 1, 2 nd 3 mg L 1 eugenol t ech of the loding densities (6 g L 1 : F 3,8 = 11.44, P <.1; 12 g L 1 : F 3,8 = 16.22, P <.1; nd 24 g L 1 : F 3,8 = 14.93, P <.1). Yellow perch exposed to 3 mg L 1 eugenol hd significntly lower metolic rtes thn the yellow perch exposed to nd 1 mg L 1 eugenol t the 6 g L 1 loding density (P <.5). Yellow perch exposed to 2 mg L 1 eugenol t the 6 g L 1 loding density showed lower men metolic rtes thn the nd 1 mg L 1 eugenol exposures, ut the difference ws not sttisticlly significnt. Metolic rtes of yellow perch t the 12 g L 1 loding density were significntly lower for the, 2 nd 3 mg L 1 eugenol exposures reltive to the 1 mg L 1 exposure (P <.5). The 24 g L 1 loding density hd significntly lower metolic rtes when exposed to 3 mg L 1 eugenol reltive to yellow perch exposed to, 1 nd 2 mg L 1 eugenol (P <.5). Overll, yellow perch exposed to 2 nd 3 mg L 1 eugenol hd lower men metolic rtes thn yellow perch exposed to nd 1 mg L 1 eugenol t ll three loding densities. An dditionl comprison ws conducted to determine the effect of loding density on metolic rte t ech eugenol concentrtion (Fig. 2). Significnt differences mong loding densities were detected for yellow perch exposed to 3 mg L 1 eugenol (F 2,6 = 7.613, P <.5). Yellow perch exposed to 3 mg L 1 eugenol t the 12 g L 1 loding density hd significntly higher metolic rtes thn yellow perch exposed to 3 mg L 1 eugenol t the 6 nd 24 g L 1 loding densities (P <.5). No significnt differences in metolic rtes were detected for yellow perch exposed to, 1 nd 2 mg L 1 eugenol. Nile tilpi 12, 24 nd 36 g L 1 loding densities The metolic rtes for Nile tilpi exposed to, 1, 2 nd 3 mg L 1 eugenol t the three Tle 2 Loding densities (SD) clculted from wet weight (g) of fish nd respirometry chmer volume (L) Loding density (g L 1 ) Yellow Perch Nile tilpi 1.1 (1.3) 9.74 (4.5) 16.2 (.7) (1.3) (7.6) 21.5 (.7) 7.95 (.2) Yellow Perch Nile tilpi 22.2 (2.7) 59.5 (2.3) (4.3) (6.9) 39. (8.7) (7.) (1.2) 39.6 (13.7) 4 Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9

5 Aquculture Reserch, 214, 1 9 Effects of eugenol on metolic rtes A R Cupp et l Control 6 1 mg L 1 2 mg L 1 Figure 1 Men mss-specific metolic rtes [mg O 2 kg 1 h 1 ( SEM rs)] for Nile tilpi (top) nd yellow perch (ottom) t three high loding densities exposed to, 1, 2 nd 3 mg L 1 eugenol. Tretments with different letters re significntly different from ech other (P <.5) Loding density (g L 1 ) 3 mg L 1 24 loding densities re shown in Fig. 1. Metolic rtes for Nile tilpi exposed to 3 mg L 1 eugenol t the 12 g L 1 loding density were significntly lower thn the metolic rtes of Nile tilpi exposed to mg L 1 eugenol t the 12 g L 1 density (F 3,8 = 5.764, P <.5). No other significnt differences in metolic rtes were oserved t the 24 or 36 g L 1 loding densities when exposed to ll four concentrtions of eugenol. Metolic rtes for Nile tilpi were compred t ech eugenol concentrtion (Fig. 2). No significnt differences in metolic rtes were detected etween the three loding densities t, 1, 2 nd 3 mg L 1 eugenol. Yellow perch lowest density Yellow perch exposed to eugenol t the two fish loding density (Fig. 3) resulted in significnt differences in metolic rtes (F 3,8 = 7.743, P <.1). The metolic rte for yellow perch exposed to 3 mg L 1 eugenol ws significntly lower thn the nd 1 mg L 1 eugenol tretments (P <.5). The 2 mg L 1 eugenol tretment resulted in decresed men metolic rte, ut the decrese ws not significntly lower thn the nd 1 mg L 1 eugenol exposures. Nile tilpi lowest density Significntly reduced metolic rtes (F 3,8 = 1.88, P <.1) were oserved for Nile tilpi exposed to 3 mg L 1 reltive to, 2 nd 2 mg L 1 eugenol exposed Nile tilpi (P <.5). The 2 mg L 1 eugenol exposure resulted in decresed metolic rte reltive to the nd 1 mg L 1 eugenol exposures, ut the decrese ws not sttisticlly significnt. Discussion Respirometry experiments re importnt indictors to understnd how fish respond to chnges in their environment (Cech 199). During events such s live trnsport, fish re sujected to confined environment for potentilly long durtions (>1 h) t loding densities greter thn typiclly encountered in most rering systems (Ross & Ross 8). The high loding densities of live trnsport Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9 5

6 Effects of eugenol on metolic rtes A R Cupp et l. Aquculture Reserch, 214, gl 1 24 gl 1 36 gl Control gl 1 12 gl 1 24 gl Control Eugenol (mg L 1 ) Figure 2 Men mss-specific metolic rtes [mg O 2 kg 1 h 1 ( SEM rs)] for Nile tilpi (top) nd yellow perch (ottom) exposed to, 1, 2 nd 3 mg L 1 eugenol t three high loding densities. Tretments with different letters re significntly different from ech other (P <.5) Control 1 mg L 1 2 mg L Nile lpi Yellow perch 3 mg L 1 Figure 3 Men mss-specific metolic rtes [mg O 2 kg 1 h 1 ( SEM rs)] for Nile tilpi nd yellow perch t the lowest loding density when exposed to, 1, 2 nd 3 mg L 1 eugenol. No interspecies rte comprison ws nlysed. Tretments with different letters re significntly different from ech other (P <.5). expose fish to indvertent stressors (Hrmon 9; Iverson, Elissen & Finstd 9) tht cn negtively impct post-trnsport survivl (Kiser & Vine 1998; Person, Smll, Beechm, Sink, LBrre & Minchew 9). Fish trnsporters would enefit, oth in efficiency nd economiclly, from sfely huling fish t higher loding densities thn re currently used in the industry without compromising fish welfre. Due to the gp of informtion on eugenol sedtion, the results of this study were compred with studies conducted using clove oil where eugenol is the primry ctive ingredient (7 9% y weight, Ross & Ross 8). Yellow perch Sufficient dissolved oxygen is primry determinnt in the success of live trnsport nd dissolved 6 Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9

7 Aquculture Reserch, 214, 1 9 Effects of eugenol on metolic rtes A R Cupp et l. oxygen levels re generlly inversely relted to fish iomss (Piper, McElwin, Orme, McCrren, Fowler & Leonrd 1982). This inverse reltionship led Ci nd Summerfelt (1992) to hypothesize tht oxygen consumption my e the limiting fctor for incresing fish iomss within tnks. Hence, this study exposed yellow perch to eugenol t the typicl loding density of 6 g L 1 nd t elevted densities of 12 nd 24 g L 1 (Crmichel, Tomsso & Schwedler 1). Results of this study indicte tht metolic rtes vry for yellow perch t ech loding density when exposed to rnge of eugenol concentrtions. In generl, s eugenol concentrtion incresed, metolic rte of yellow perch decresed. Decresed oxygen consumption hs lso een documented with incresed concentrtions of sedtives 2-phenoxyethnol (Guo, Teo & Chen 1995), isoeugenol (Forgn & Forster 21) nd clove oil (Hoskonen & Pirhonen 4). However, this is the first report of dose-dependent chnge in the metolic rte of high densities of freshwter fish. Eugenol exposure t 1 mg L 1 did not significntly decrese metolic rtes reltive to the control, conversely higher metolic rte ws oserved in eugenol exposed fish thn the control t the 12 g L 1 loding density. Results of severl previous studies suggested tht concentrtions less thn 1 mg L 1 were effective to reduce the metolic rte of fish. For exmple, mg L 1 clove oil effectively reduced oxygen demnd in lrgemouth ss Micropterus slmoides most likely ecuse crdiovsculr output ws reduced (Cooke et l. 4). The cortisol stress response of mtrinx Brycon cephlus ws reduced with 5 mg L 1 clove oil when sujected to live trnsport, which would lso decrese oxygen demnd (Inoue, Afonso, Iwm & Mores 5). In ddition, Iverson et l. (9) mesured the stress response of Atlntic slmon Slmo slr smolts exposed to clove oil during trnsport to se nd found tht clove oil s low s 4. mg L 1 ws successful in reducing mortlity. Results from this study suggest tht higher eugenol concentrtion (i.e. 2 mg L 1 eugenol) is needed to induce reduction in metolic rtes for yellow perch t elevted loding densities. In ddition, to consistently decrese metolic rtes, yellow perch should e exposed to 3 mg L 1 eugenol for loding densities up to 24 g L 1. The lower sedtive concentrtions from the previous studies cn potentilly e ttriuted to their: (1) lower loding densities, (2) different physiologicl responses monitored, (3) interspecies differences, (4) dditionl ctive constituents of clove oil nd (5) vrying tempertures. The lowest loding density ( g L 1 ) ws much lower thn typicl trnsport density in production quculture (Crmichel et l. 1). The significnt reduction in metolic rtes when exposed to 3 mg L 1 eugenol is importnt for understnding generl sedtion respirtory responses nd iding in low density trnsport (e.g. short durtion trnsports <1 h, reserch purposes nd intr-htchery opertions). The similrity etween reduced metolic rtes t the elevted loding densities nd this light loding density lso suggested tht the eugenol concentrtion nd not the loding density lter the metolic rtes of yellow perch exposed to eugenol. Nile tilpi Nile tilpi t loding densities higher thn currently recommended (i.e. 24 nd 36 g L 1 ) showed no significnt differences in metolic rtes when exposed to eugenol. Deriggi et l. (6) oserved no net ion imlnce in juvenile Nile tilpi exposed to 5 mg L 1 eugenol for 1 min, ut they hypothesized tht oxygen det my hve occurred during sedtion ecuse of suppressed metolic rte. By contrst, eugenol (1 3 mg L 1 ) t 24 nd 36 g L 1 loding densities my not induce the sme physiologicl response s oserved y Deriggi et l. (6) due to the lck of differences in metolic rtes. Simoes, Lomrdi, Gomide nd Gomes (211) exposed juvenile Nile tilpi to rnge of clove oil concentrtions in plstic gs t loding density of ~22 g L 1 nd found significnt effluxes in ions nd incresed mortlity for Nile tilpi t the highest concentrtion tested (18 mg L 1 clove oil). While those results differ from our findings nd those of Deriggi et l. (6), the fish tested y Simoes et l. (211) were much smller ( g) nd were sedted for sustntilly longer durtion (24 h). Physiologicl chnges resulting from the effects of sedtion hve een found to e more pronounced in erly life stge fish (Hoskonen & Pirhonen 4). Significntly lower metolic rtes were oserved for Nile tilpi exposed to 3 mg L 1 eugenol reltive to controls ( mg L 1 eugenol) t the Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9 7

8 Effects of eugenol on metolic rtes A R Cupp et l. Aquculture Reserch, 214, g L 1 loding density, loding density tht is commonly used in Nile tilpi trnsport. Nile tilpi held t this typicl density my enefit from sedtion using 3 mg L 1 eugenol due to the reduction in metolic rtes (Crmichel et l. 1). Nile tilpi exposed to eugenol t the lowest loding density ( g L 1 ) showed significntly lower metolic rtes, when exposed to 3 mg L 1 eugenol reltive to ll other eugenol concentrtions. Although no significnt differences in metolic rtes were found t the 24 nd 36 g L 1 loding densities for Nile tilpi exposed to 3 mg L 1 eugenol, the significntly lower metolic rtes found t the 39 nd 12 g L 1 loding densities suggest tht eugenol my e more effective t reducing metolic rtes t lower loding densities (e.g. generl hndling nd sedtion). Conclusion Higher concentrtions of eugenol thn previously reported my e required to reduce metolic rtes when fish re held t high loding densities. Yellow perch t loding densities up to 24 g L 1 hd reduced metolic rtes when exposed to 2 nd 3 mg L 1 eugenol reltive to the control. Exposure to 3 mg L 1 eugenol lso led to vrile metolic rtes in yellow perch. Nile tilpi exposed to 3 mg L 1 eugenol t loding density of 12 g L 1 hd reduced metolic rtes reltive to the control. Loding densities greter thn 12 g L 1 did not hve reduced metolic rtes when exposed to eugenol. The results of this reserch suggest tht eugenol cn lower metolic rtes of different species held t high loding densities. Specificlly, exposure of yellow perch to eugenol my result in reduced metolic rtes t current or incresed loding densities while the effectiveness of eugenol to reduce metolic rtes of Nile tilpi ppers to e more effective t lower loding densities. Acknowledgments We thnk United Sttes Deprtment of Agriculture North Centrl Regionl Aquculture Center for providing project funding nd AQUI-S New Zelnd, Ltd. for supplying test chemicl. Any use of trde, firm or product nmes is for descriptive purposes only nd does not imply endorsement y the US Government. References Adel-Mgid A.M. & Biker M.M. (1975) Oxygen consumption nd respirtory ehvior of three Nile fishes. Hydroiologi 46, Anderson W.G., McKinley R.S. & Colvecchi M. (1997) The use of clove oil s n nesthetic for rinow trout nd its effects on swimming performnce. North Americn Journl of Fisheries Mngement 17, Brton B.A. & Iwm G.K. (1991) Physiologicl chnges in fish from stress in quculture with emphsis on the response nd effects of corticosteroids. Annul Review of Fish Diseses 1, Bemish F.W. (1963) Respirtion in fishes with specil emphsis on stndrd oxygen consumption. Cndin Journl of Zoology 42, Bowker J. & Trushenski J. (212) Hedliners. Fisheries 37, Ci Y. & Summerfelt R.C. (1992) Effects of temperture nd size on oxygen consumption nd mmoni excretion y wlleye. Aquculture 14, Crmichel G.J., Tomsso J.R. & Schwedler T.E. (1) Fish trnsport. In: Fish Htchery Mngement (ed. y G.A. Wedemeyer), pp Americn Fisheries Society, Bethesd, MD. Crneiro P.C., Kiseler P.H., Swrofsky E. & Bldisserotto B. (9) Trnsport of jundi juveniles t different loding densities, wter qulity nd lood prmeters. Neotropicl Ichthyology 7, Cech J.J. (199) Respirometry. In: Methods for Fish Biology (ed. y C.B. Schreck & P.B. Moyle), pp Americn Fisheries Society, Bethesd, MD. Cho G.K. & Heth D.D. () Comprison of tricine methnesulphonte (MS-222) nd clove oil nesthesi effects on the physiology of juvenile Chinook slmon Oncorhynchus tshwytsch. Aquculture Reserch 31, Cooke S.J., Suski C.D., Ostrnd K.G., Tufts B.L. & Whl D.H. (4) Behviorl nd physiologicl ssessment of low concentrtions of clove oil nesthetic for hndling nd trnsporting lrgemouth ss Micropterus slmoides. Aquculture 239, De Areu J.S., Snri-Ocho A.I., Gonclves F.D. & Urinti E.C. (8) Stress responses of juvenile mtrinx fter trnsport in closed system under different loding densities. Cienci Rurl 38, Deriggi G.F., Inoue L.A. & Mores G. (6) Stress responses to hndling in Nile tilpi Oreochromis niloticus: ssessment of eugenol s n lterntive nesthetic. Act Scientrium Biologicl Sciences 28, El-Syed A.F. (6) Tilpi Culture. CABI Pulishing, Cmridge, MA. FAO (9) Fishery nd quculture sttistics: quculture production. Aville from: docrep/16/q187t/q187t.pdf (ccessed 18 June 213). 8 Pulished 214. This rticle is U.S. Government work nd is in the pulic domin in the USA., Aquculture Reserch, 1 9

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